The advent of Polymerase Chain Reaction (PCR) since the mid 1980s has revolutionized molecular biology through vastly extending the capability to identify, manipulate, and reproduce DNA. Nowadays PCR is routinely practiced in the course of conducting scientific researches, clinical diagnostics, forensic identifications, and environmental studies. Polymerase chain reaction (PCR) is a template-directed polymerization reaction that provides a method for amplifying specific nucleic acids in vitro. PCR can produce a million to a billion fold copies of a DNA template in a single enzymatic reaction within a matter of minutes to hours, enabling researchers to determine the size and sequence of a target DNA.
Despite significant amount of progress made in the field of molecular biology and engineering, PCR remains a challenging application. One particular challenge is non-specific DNA amplification due to mis-priming, where the set of PCR primers hybridize to non-target DNA sequences. In principle, specific hybridization of a primer to the target DNA should ensure amplification of the target DNA and not other contaminating sequences. Specific priming can mostly be achieved at higher temperatures, such as at about 60° C.-70° C. These are also the temperatures at which the DNA polymerase Taq is most active. Thus, the chain extension reaction is typically set to take place at these temperatures to maximize specific DNA amplification. However, at a lower temperature, such as at room temperature, where components of PCR reactions are assembled, mis-priming occurs more frequently. Mis-priming may result from partial hybridization between the forward and reverse primers (i.e., primer-dimer formation), or from hybridization between a primer and a partially complementary sequence in the DNA template. These weakly formed hybrids may be sufficiently stable at or around room temperature. Because Taq still exhibits significant, though not optimal, activity at room temperature, the nonspecific hybrids can lead to nonspecific PCR products, which act as templates for even greater amount of nonspecific product formation during PCR reaction. In some cases, such as in the case of low template copy number, nonspecifically amplified products can be the dominant PCR products.
To overcome the aforementioned problem, various so-called “hot-start” PCR methods have been developed to suppress non-specific amplification at a temperature below the usual operating PCR temperatures. In general, most of the known hot-start methods employ a hot-start polymerase or polymerase complex that has no or very low activity at a temperature below the usual operating PCR temperatures. For example, polymerase-specific monoclonal antibodies have been developed to inhibit the enzyme activity by forming a polymerase-antibody enzyme complex. At room temperature, the enzyme complex is stable and thus inactive. When the temperature is raised to above 90° C., the enzyme complex releases the antibodies and thus becomes activated (U.S. Pat. No. 5,338,671, Scalice E R et al, Kellogg et al, (1994) Biotechniques 16:1134-1137). To sufficiently suppress the enzyme activity at room temperature, a high an antibody to enzyme molar ratio (typically 1 to 7 folds) is often required. The high antibody to enzyme ratio makes this method relatively costly. Another drawback is that the enzyme-antibody binding is reversible to some degree. Thus, even though the enzyme activity is significantly higher at the operating PCR temperatures, the enzyme may not be fully activated, compared to the enzyme without the presence of the antibodies.
Another approach to develop a hot-start polymerase is to chemically modify the lysine residues of the enzyme using a heat-sensitive modifying group. The modified enzyme is inactive at below the PCR temperature. However, once the modified enzyme is heated to a higher temperature, such as at above 90° C., the modifying group is released from the enzyme, thereby activating the enzyme (U.S. Pat. Nos. 5,677,152 and 6,183,998). This method has two attractive features. The first one is that both the modifying chemical and the manufacturing process for the modified enzyme are simple and inexpensive. The second feature is that the enzyme activation is, in principle, an irreversible process. Therefore, potentially a greater amount of enzyme activity can be achieved on activation, compared to the antibody-based hot-start enzyme. In practice, however, the conventional hot-start DNA polymerases that are chemically modified suffer from a number of profound drawbacks. Chemically modified hot-start enzymes are slow to start. For example, AmpliTaq Gold, a commercially available chemically-modified Taq, may require an activation time of as long as 15-20 minutes, which makes up a significant portion of the overall PCR time. Chemically modified Taq is also known to degrade significantly during storage as often indicated by progressively lower recovery activation efficiency. Moreover, following heat activation, chemically modified Taq in general does not recover the full enzyme capacity of the corresponding unmodified enzyme, even if the modified enzyme is freshly prepared.